Tetrahydrofurfuryl alcohol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

not reported

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

The Japanese relevant authority has stated clearly in secondary summaries that this study was conducted in compliance with GLP. However, some pages, which may contain the confirmation of GLP compliance, are missing from the published study report. Overall, the studies are the part of safety evaluation by the national authority of Japan (from Japan Existing Chemical Data Base (JECDB)). The data are collected under the control of the authority and approved by the GLP authority, which are under national control. Therefore we consider that the studies are in compliance with GLP although GLP or QA statement is not included in the available study reports.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

2AA- only control with metabolic activation

GLP compliance:

yes

Remarks:

evidence of GLP is not included in the report

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Tetrahydrofurfuryl alcohol

EC Number:

202-625-6

EC Name:

Tetrahydrofurfuryl alcohol

Cas Number:

97-99-4

Molecular formula:

C5H10O2

IUPAC Name:

tetrahydrofuran-2-ylmethanol

Details on test material:

Name of test substance; Tetrahydrofurfuryl alcohol
Structural formula; see attachment
Molecular weight; 102.13
Purity; 99.5%
Impurity; 5-Methyltetrahydrofurfuryl alcohol (GC): 0.34%
Appearance under ambient temperature; transparent liquid
Substance type; Pure active substance
Lot. No.; not reported

Method

Target gene:

S. typhimurium TA100 and TA1535; his G (base-pair substitution)
S. typhimurium TA98; his D (frameshift)
S. typhimurium TA 1537; his C (frameshift)
E. coli WP2uvrA/pK101; trp E (base-pair substitute)

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital and tetrahydrofurfuryl alcohol induced rat liver S9

Test concentrations with justification for top dose:

Tester strains; TA 100, TA1535, WP2uvrA/pK101, TA 98 and TA 1537: 5000, 2500, 1250, 625 and 313 µg/plate (with or without S9 mix)
Positive substance;
with S9 mix: TA 100 (2-aminoanthracene (2-AA); 1 µg/plate), TA1535 (2-AA; 2 µg/plate), WP2uvrA/pK101 (2-AA; 2 µg/plate), TA 98 (2-AA; 0.5 µg/plate) and TA 1537 (2-AA; 2 µg/plate)
without S9 mix: TA 100 (furfurylamide (AF-2); 0.01 µg/plate), TA1535 (sodium azide (NaN3); 0.5 µg/plate), WP2uvrA/pK101 (ENNG; 2 µg/plate), TA 98 (AF-2; 0.5 µg/plate) and TA 1537 (9-AA; 2 µg/plate)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Water for injection (Otsuka Pharmaceutical Factory, Inc., Lot No.; K0C78)
- Justification for choice of solvent/vehicle: The test substance was soluble in water for injection (DW) at the concentration of 50 mg/ml in the preliminary test for selection of solvent. No exothermic, bubbling and discoloration was observed when DW was added. Based on these results, DW was used for solvent.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

furylfuramide

Remarks:

AF-2 (Lot No.; CAP0185, content; 98.9%, Wako Pure Chemical Industries, Ltd.), Solvent: DMSO

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

NaN3 (Lot No.; KWE6685, content; 96.5%, Wako Pure Chemical Industries, Ltd.), Solvent: DW

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

ENNG (Lot No. 56F-3651, content; 99.0%, Sigma Chemical Company), Solvent: DMSO

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DW

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

9-AA (Lot No., 80F-0186, content; >99%, Sigma Chemical Company), Solvent: DMSO

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2-AA)

Remarks:

2-AA (Lot No. TWH2355, content; 98.0%, Wako Pure Chemical Industries, Ltd.), Solvent: DMSO

Details on test system and experimental conditions:

1. Tester strain;
Five tester strains, Salmonella typhimurium strains TA100, TA1535, TA98 and TA1537 which were obtained from the University of California on May 27, 1983 and Escherichia coli strain WP2uvrA/pKM101 which was obtained from Japan Bioassay Research Center were used. These strains are widely used for bacterial reverse mutation test, and are recommended by OECD Guidelines and the guideline in Japanese Chemical Substances Control Law.
2. Bacterial suspension;
Each frozen tester strains were thawed at use, and aliquot of 20 µl was taken and inoculated to 10 µl of liquid complete medium. They were incubation under shaking culture for 8 hours at 37°C (90 agitation/min.). L-shaped tube (capacity; 22 ml) was used for culturing vessel. After incubation period was finished, turbidity of bacterial suspension was measured with turbidity meter. Viable bacteria count was calculated by conversion from turbidity. The appropriate bacterial concentration (10 E+09/ml or more) was confirmed in the suspension and then used for the test (Note 2).
3. Reverse mutation test;
The test was conducted by pre-incubation method with and without S9 mix. For each dosage, 0.1 ml of test substance solution or negative (solvent) control material was added into sterilized tube, and then 0.5 ml of 0.1 mol/l sodium-phosphate buffer (pH 7.4) (without S9 mix) or 0.5 ml of S9 mix (with S9 mix) was added. Then 0.1 ml of bacterial suspension was added and was shaking-incubated at 37°C for 20 minutes. After pre-incubation, 2 ml of top agar was added to the mixture and was multilayered onto minimum glucose agar plate medium. After solidification of multilayered top agar, it was incubated at 37°C for 48 hours. Growth of bacterial flora was observed with stereoscopic microscope to investigate degree of bacterial growth inhibition by test material and then the presence of precipitation was investigated by visual inspection. Number of revertant colonies on the plate was counted with automatic colony counter (CA-11, System Science Co., Ltd.; correction method: area and miscount correction). Tests for the positive control materials were conducted similarly.

Evaluation criteria:

If more than 2-fold increase is noted in the number of revertant colonies (mean value) compared with negative (solvent) control with our without S9 mix in any tester strain and also if reproducibility is noted in such increase, test substance is judged to have mutagenicity (positive). For other case, it is judged as negative.

Statistics:

No statistical method was used for judgment of test results.

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The numbers of revertant colonies were below 2 fold of negative (solvent) control value at the dosages of 5000, 1250, 313, 78.1, 19.5, 4.88 and 1.22 µg/plate for all tester strains with or without S9 mix as the result of the preliminary test. No growth inhibition and no precipitation were observed for any tester strain with or without S9 mix.
Based on above results, main tests were conducted at 5 dosages of 5000, 2500, 1250, 625 and 313 µg/plate for all tester strains.
As the result of 2 main tests, the numbers of revertant colonies for all tester strains were below 2 folds of negative (solvent) control value with or without S9 mix. No growth inhibition and no precipitation were observed for any tester strain with or without S9 mix.
From sterility tests for the test substance solution at the highest concentration and S9 mix, no contamination by bacteria or mould which affected the results of the tests was observed.
Based on the results of preliminary test, main tests were conducted with the highest concentration of 5000 µg/plate. 
Number of revertant colonies was below 2 folds of negative (solvent) control value for all tester strains with
or without S9 mix.
It was confirmed that the test was appropriately conducted because negative (solvent) control value and positive
control value in this test are within the normal range which calculated from historical background data, and because
positive control material showed positive result with significant increase of over 2 fold in the number of revertant
colonies which was induced by the positive control material in each tester strains with or without S9 mix compared
with the number of negative (solvent) control in each tester strains.
Based on above results, it was concluded that tetrahydrofurfuryl alcohol has no mutagenicity (negative) for
bacterial reverse mutation test.

Any other information on results incl. tables

Pre-experiment for toxicity

Whether or not there is metabolic activation		Dosage level of the test substance (µg/plate)	Number of reverse mutations (number of colonies/plate)
			Base pair substitution type			Frame shift type
			TA100	TA1535	WP2uvrA/pKM101	TA98	TA1537
－S9 mix		Negative control	114	9	81	16	17
		1.22	130	10	87	23	23
		4.88	109	8	84	24	24
		19.5	123	13	67	26	29
		78.1	116	14	71	24	24
		313	126	10	83	18	22
		1250	118	8	65	27	24
		5000	105	13	73	26	25
+S9 mix		Negative control	113	8	78	32	24
		1.22	117	10	86	26	31
		4.88	126	14	114	35	20
		19.5	120	11	112	25	24
		78.1	128	8	89	34	30
		313	123	9	125	39	22
		1250	114	8	104	32	27
		5000	108	11	88	33	25
Positive control	Positive control groups for which  S9 mix is not necessary	Name	AF-2	NaN3	ENNG	AF-2	9-AA
		Dosage (µg/plate)	0.01	0.5	2	0.1	80
		(Number of colonies/plate)	551	467	3659	697	263
	Positive control groups for whichS9mix is necessary	Name	2-AA	2-AA	2-AA	2-AA	2-AA
		Dosage (µg/plate)	1	2	2	0.5	2
		(Number of colonies/plate)	1298	182	625	400	184

Results of main experiment 1, preincubation; revertants per plate (mean and s.d.)

Whether or not there is metabolic activation		Dosage level of test substance (µg/plate)	Number of reverse mutations (number of colonies/plate)
			Base pair substitution type						Frame shift type
			TA100		TA1535		WP2uvrA/ pKM101		TA98		TA1537
－S9 mix		Negative control	103 123 113	(113) (± 10)	14 11 11	(12) (± 2)	82 75 87	(81) (± 6)	22 20 22	(21) (± 1)	17 16 17	(17) (± 1)
		313	97 103 122	(107) (± 13)	11 8 10	(10) (± 2)	93 73 92	(86) (± 11)	17 23 21	(20) (± 3)	16 21 15	(17) (± 3)
		625	112 122 108	(114) (± 7)	10 8 12	(10) (± 2)	76 76 98	(83) (± 13)	21 20 19	(20) (± 1)	15 18 14	(16) (± 2)
		1250	110 134 96	(113) (± 19)	14 11 10	(12) (± 2)	63 77 95	(78) (± 16)	31 18 25	(25) (± 7)	18 20 15	(18) (± 3)
		2500	116 109 111	(112) (± 4)	11 9 9	(10) (± 1)	93 81 92	(89) (± 7)	20 17 24	(20) (± 4)	17 11 17	(15) (± 3)
		5000	118 119 102	(113) (± 10)	10 16 11	(12) (± 3)	87 70 91	(83) (± 11)	17 21 17	(18) (± 2)	17 23 15	(18) (± 4)
+S9 mix		Negative control	128 127 102	(119) (± 15)	13 8 15	(12) (± 4)	99 106 99	(101) (± 4)	33 24 23	(27) (± 6)	25 24 22	(24) (± 2)
		313	114 120 119	(118) (± 3)	9 11 11	(10) (± 1)	87 111 92	(97) (± 13)	40 23 29	(31) (± 9)	26 27 21	(25) (± 3)
		625	137 98 125	(120) (± 20	13 10 12	(12) (± 2)	92 102 77	(90) (± 13)	33 30 23	(29) (± 5)	27 23 22	(24) (± 3)
		1250	108 126 106	(113) (± 11)	12 11 17	(13) (± 3)	104 111 112	(109) (± 4)	30 31 37	(33) (± 4)	22 27 24	(24) (± 3)
		2500	113 120 123	(119) (± 5)	10 16 15	(14) (± 3)	109 95 112	(105) (± 9)	29 29 33	(30) (± 2)	26 24 18	(23) (± 4)
		5000	101 120 113	(111) (± 10)	10 18 13	(14) (± 4)	86 113 116	(105) (± 17)	23 33 23	(26) (± 6)	25 30 29	(28) (± 3)
Positive control	Positive control groups for which  S9 mix is not necessary	Name	AF-2		NaN3		ENNG		AF-2		9-AA
		Dosage (µg/plate)	0.01		0.5		2		0.1		80
		(Number of colonies/plate)	564 555 533	(551) (± 16)	520 476 468	(488) (± 28)	4412 4259 3992	(4221) (± 213)	632 644 625	(634) (± 10)	292 237 346	(292) (± 55)
	Positive control groups for whichS9mix is necessary	Name	2-AA		2-AA		2-AA		2-AA		2-AA
		Dosage (µg/plate)	1		2		2		0.5		2
		(Number of colonies/plate)	1381 1446 1443	(1423) (± 37)	169 149 171	(163) (± 12)	935 955 925	(938) (± 15)	436 437 451	(441) (± 8)	171 167 175	(171) (± 4)

AF-2:2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

NaN₃: Sodium azide

ENNG:N-ethyl-N’-nitro-N-nitrosoguanidine,

9-AA:9-Aminoacridine hydrochloride,

2-AA:2-Aminoanthracene

Results of main experiment 2, preincubation; revertants per plate (mean and s.d.)

Whether or not there is metabolic activation		Dosage level of test substance (µg/plate)	Number of reverse mutations (number of colonies/plate)
			Base pair substitution type						Frame shift type
			TA100		TA1535		WP2uvrA/ pKM101		TA98		TA1537
－S9 mix		Negative control	121 118 102	(114) (± 10)	16 15 14	(15) (± 1)	82 85 88	(85) (± 3)	19 22 22	(21) (± 2)	18 14 11	(14) (± 4)
		313	127 111 108	(115) (± 10)	14 11 12	(12) (± 2)	96 94 86	(92) (± 5)	24 24 26	(25) (± 1)	17 16 15	(16) (± 1)
		625	95 106 107	(103) (± 7)	9 10 10	(10) (± 1)	95 86 89	(90) (± 5)	16 24 25	(22) (± 5)	16 16 15	(16) (± 1)
		1250	110 112 117	(113) (± 4)	10 11 12	(11) (± 1)	76 90 88	(85) (± 8)	17 25 21	(21) (± 4)	14 18 17	(16) (± 2)
		2500	101 105 105	(104) (± 2)	13 9 15	(12) (± 3)	86 90 91	(89) (± 3)	16 17 20	(18) (± 2)	15 13 17	(15) (± 2)
		5000	121 98 130	(116) (± 17)	8 11 14	(11) (± 3)	93 87 81	(87) (± 6)	26 18 23	(22) (± 4)	12 10 12	(11) (± 1)
+S9 mix		Negative control	110 127 108	(115) (± 10)	11 11 16	(13) (± 3)	93 95 87	(92) (± 4)	32 29 35	(32) (± 3)	23 23 15	(20) (± 5)
		313	123 114 126	(121) (± 6)	12 13 13	(13) (± 1)	88 111 91	(97) (± 13)	24 31 27	(27) (± 4)	20 17 22	(20) (± 3)
		625	124 119 110	(118) (± 7)	14 16 14	(15) (± 1)	90 87 103	(93) (± 9)	39 21 24	(28) (± 10)	18 22 29	(23) (± 6)
		1250	121 115 112	(116) (± 5)	10 13 10	(11) (± 2)	87 95 102	(95) (± 8)	25 28 33	(29) (± 4)	22 25 22	(23) (± 2)
		2500	108 99 116	(108) (± 9)	14 10 13	(12) (± 2)	90 96 108	(98) (± 9)	24 27 30	(27) (± 3)	22 16 20	(19) (± 3)
		5000	108 114 102	(108) (± 6)	9 13 10	(11) (± 2)	101 87 108	(99) (± 11)	25 25 28	(26) (± 2)	22 19 19	(20) (± 2)
Positive control	Positive control groups for which  S9 mix is not necessary	Name	AF-2		NaN3		ENNG		AF-2		9-AA
		Dosage (µg/plate)	0.01		0.5		2		0.1		80
		(Number of colonies/plate)	563 520 482	(552) (± 41)	369 387 440	(399) (± 37)	4358 4439 4275	(4357) (± 82)	632 618 590	(613) (± 21)	242 209 209	(220) (± 19)
	Positive control groups for whichS9mix is necessary	Name	2-AA		2-AA		2-AA		2-AA		2-AA
		Dosage (µg/plate)	1		2		2		0.5		2
		(Number of colonies/plate)	1398 1398 1301	(1366) (± 56)	150 147 138	(145) (± 6)	1347 1373 1072	(1264) (± 167)	432 428 423	(428) (± 5)	200 182 194	(192) (± 9)

Applicant's summary and conclusion

Conclusions:

Tetrahydrofurfuryl alcohol has been tested according to a Japanese national method that is equivalent to OECD TG 471. No evidence of test-substance induced increases in the number of revertants was obtained using S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2, with and without metabolic activation, in either the initial or the repeat experiment, both of which used the pre-incubation method. Appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.